Galectin-3 plasmapheresis therapy

ABSTRACT

The invention is directed to the removal of serum gal-3 from circulation by plasmapheresis, comprising at least in part donor apheresis, using gal-3 binding agents in either a fixed bed, or in a form easily removed, such as by being complexed with magnetic particles. This method, on its own, brings a sharp reduction and relief from the inflammation and fibroses that can be induced by circulating gal-3. The process may be combined with the administration of gal-3 binding agents, such as modified citrus pectin, to further lower unbound gal-3 levels, to the point where gal-3 in the tissues may be addressed. This method may also be combined with removal of TNF receptors to provide an effective treatment for cancer.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/629,932, filed Sep. 28, 2012 and is related to U.S.Provisional Patent Application No. 61/568,210 filed Dec. 8, 2011, bothof which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to treatment of disease and biologicalconditions mediated at least in part by one or more galectins. Galectinsare a family of lectins (sugar binding proteins) that are characterizedby having at least one carbohydrate recognition domain (CRD) with anaffinity for beta-galactosides. These proteins were recognized as afamily only recently, but are found throughout the animal kingdom, andare found in mammals, birds, amphibians, fish, sponges, nematodes andeven fungi. This application focuses on galectins in mammals, and inparticular, humans. Although the invention herein may be employed withboth companion animals (e.g., pets such as dogs and cats) and commercialanimals (such as cows, pigs and sheep) the methods and subject matteraddressed herein are particularly focused on the treatment of humans.

Galectins mediate and modulate a wide variety of intracellular andextracellular functions, and thus are both expressed within the cell andfrequently targeted to a specific cytosolic site, and secreted from thecell, for distribution extra-cellularly, as a component of human plasma.Among the many functions that are mediated by extracellular galectinsare inflammation, fibrosis formation, cell adhesion, cell proliferation,metastatic formation, angiogenesis (cancer) and immunosuppression.

Galectins are a family of fifteen (15) carbohydrate-binding proteins(lectins) highly conserved throughout animal species. Most galectins arewidely distributed, though galectin -5, -10 and -12 show tissue-specificdistribution. While galectins are variably expressed by all immunecells, they are upregulated in activated B and T cells, inflammatorymacrophages, natural killer (NK) cells, and FoxP3 regulatory T cells.Galectins contain a variety of structural arrangements, but a relativelyconserved carbohydrate recognition domain (CRD). The majority ofgalectins display a single CRD, and are biologically active as monomers(galectin-5, -7 and -10), or require homodimerization for functionalactivity (galectin-1, -2, -11, -13, -14 and -15). Alternatively,tandem-repeat-type galectins (galectin-4, -8, -9, and -12) contain twoCRDs separated by a short linker peptide, while galectin-3 (chimerictype) has a single CRD fused to a non-lectin domain that can becomplexed with other galectin-3 monomers to form an oligomeric pentamer.Of note, some galectins, such as galectin-10, bind to mannose-containingglycans. Among the family of galectins, -1, -3, and -9 are particularlyimportant as potential therapeutic targets, and -2, -4, -5, -6, -7, -8,-10, -11, -12, -13, -14, and -15 also appear implicated in a variety ofbiological pathways associated with morbidity and mortality.

Thus, galectin-7 has been implicated in the development of certain formsof cancer. St. Pierre et al, Front. Biosci., 1:17, 438-50 (2012) and ina variety of specific cancers, including gal-2, -4 and -8 in the contextof colon and breast cancer, Barrow et al, Clin. Cancer Res., 15;17 (22)7035-46 (2011). Squamous cell carcinoma of the tongue, Alves et al.,Pathol. Res. Pract. 15;207 (4) 236-40 (2011) has been shown to beassociated with elevated levels of gal-1, -3 and -7, while cervicalsquamous carcinoma has been shown linked to gal-7 levels, Zhu et al,Int. J. Cancer, (August, 2012). A number of galectins, including gal-15,gal-13 and gal-10 have been demonstrated to be linked to implantationand pregnancy concerns. See, e.g., Than et al, Eur. J. Biochem. 271 (6)1065-78 (2004), Lewis et al, Biol. Reprod. 77 (6); 1027-36 (2007). Anumber of galectins, including gal-2, 3, 8 and others have beenidentified as correlating with various autoimmune disorders, such aslupus. Salwati et al, J. Infect. Dis. 1;202 (1) 117-24 (2010), Pal etal, Biochim. Biophys. Acta., 1820 (10) 1512-18 (2012) and Janko et al,Lupus 21 (7):781-3 (2012). Elevated levels of a number of galectins,including gal-3, are associated with inflammation and fibrosesencountered in wound healing and the like. Gal et al, Acta. Histochem.Cytochem. 26:44 (5); 191-9 (2011).

Quite obviously, mediation of inflammatory and fibrotic pathways makesgalectins critical elements of a wide variety of disease, injury andtrauma related phenomena. In many cases, the presence of unwantedconcentrations of galectins can aggravate a disease condition or traumasituation, or interfere with attempts to treat diseases, such as canceror congestive heart failure. Among the family of galectins recognized asactive in humans, galectin-1, galectin-3 and galectin-9 are ofparticular interest. As indicated above, these proteins are generallyreferred to, and referred to herein as, gal-1, gal-3 and gal-9. A widevariety of conditions in humans, ranging from problems in conceiving toasthma to chronic heart failure to cancer to viral infection to strokeand beyond are mediated or aggravated by higher than normalconcentrations of galectins. Thus, among other galectins, gal-3 isparticularly prominent in fibrosis, inflammation and cell proliferation,while gal-1 also plays a role in the immunosuppression required for asuccessful pregnancy. Gal-1 is also thought to be involved in thedifferentiation of nerve cells. Gal-9 has been shown to be involved inthe control of lesions arising from immunoinflammatory diseases, and isgenerally implicated in inflammation—gal-9 apparently plays a role ineosinophil recruitment in inflammatory sites. It also appears to mediateapoptosis in certain activated cells.

While the discussion herein is applicable to circulating active gal-1,gal-3 and gal-9, and galectins in general, where elevated circulatinggalectin levels are associated with disease or injury conditions, morehas been elucidated about the role of gal-3 in disease and traumaprogression than any of the other galectins, and so it is exemplifiedherein. More specifically, this invention focuses on the removal ofactive gal-3 from mammalian, particularly human, plasma. Gal-3 has beenshown to be involved in a large number of biological processes, many ofwhich are related to disease states of various kinds. Binding andblocking activity of gal-3 in the circulation, or removal of largeamounts of gal-3 from circulation may therefore improve existing medicaltreatments, suppress and/or reduce inflammation and fibrosis resultingfrom others, and make it possible to intervene in various disease statesnot otherwise easily treated. The invention is equally applicable to thereduction in circulating levels of other active galectins to addressconditions mediated by those galectins. By “active” galectins, what isreferred to is biologically active molecules. As noted, for example,gal-3 can be active, that is, mediate mammalian responses to varioustraumas and conditions, as a monomer and as an oligomer. In any mammal,at any given time, significant amounts of gal-3 and other galectins arepresent in an inactive state—that is, they are either tissue bound orligand bound in such fashion as to inhibit molecular interaction. Whilesuch galectins molecules may become active, and may be or become thetarget of removal by the invention disclosed herein, when monitoringpatient conditions and controlling responses, the focus of the inventionis the removal of active galectins from the blood stream. This inventionmakes use of plasmapheresis, sometimes referred to as therapeutic plasmaexchange, to control levels of gal-3, and more specifically biologicallyactive galectin, in circulation. Plasma is lead through a fluid pathwayand either intermixed with a gal-3 binding agent which can be separatedfrom the plasma, or returned to the body with blocked inactivated gal-3,or lead past a solid support which binds gal-3, the plasma beingsubsequently returned to the body with a reduced level of gal-3. Thus,this invention can be used to remove bound gal-3 as part of a strategyto reduce total gal-3 content. The focus, in this application, however,is to remove active or unbound gal-3 as a therapeutic measure.

2. Related Art

This application is related to U.S. patent application Ser. No.13/153,648, filed Jun. 6, 2011. That application in turn claims prioritybenefit to U.S. patent application Ser. No. 11/485,955, filed Jul. 6,2006. The content of both these patent applications is expresslyincorporated herein-by-reference. In U.S. patent application Ser. No.13/153,648 (U.S. Patent Publication US-2011-0294755 A1) a method oftreating cell proliferation conditions, inflammation and aggravatedfibroses is disclosed which involves the administration of an agent thatcan bind circulating gal-3, such as modified citrus pectin, (MCP), acitrus pectin which has a reduced molecular weight of twenty thousand(20,000) Daltons or less, preferably ten thousand (10,000) Daltons orso. MCP is available commercially from EcoNugenics of Santa Rosa, Calif.and is discussed in U.S. Pat. Nos. 6,274,566 and 6,462,029.

BACKGROUND OF THE TECHNOLOGY

Gal-3 is approximately 30 kDa and, like all galectins, contains acarbohydrate-recognition-binding domain (CRD) of about one hundredthirty (130) amino acids that enable the specific binding ofβ-galactosides. Gal-3 is encoded by a single gene, LGALS3, located onchromosome 14, locus q21-q22. This protein has been shown to be involvedin a large number of biological processes. The list set forth herein isexemplary only as new situations and roles for gal-3 are continuallybeing revealed. Among the biological processes at the cellular levelthat have been shown to be mediated, at least in part, by gal-3, arecell adhesion, cell migration, cell invasion, cell activation andchemoattraction, cell growth and differentiation, cell cycle,angiogenesis and apoptosis.

Given gal-3's broad biological functionality, it has been demonstratedto be involved in a large number of disease states or medicalimplications. Studies have also shown that the expression of gal-3 isimplicated in a variety of processes associated with heart failure,including myofibroblast proliferation, fibrogenesis, tissue repair,inflammation, and ventricular and tissue remodeling. Elevated levels ofgal-3 in the blood have been found to be significantly associated withincreased morbidity and mortality. They have also been found to besignificantly associated with higher risk of death in both acutedecompensated heart failure and chronic heart failure populations.

Various investigations have shown elevated levels of gal-3 to aggravatea wide variety of disease conditions associated with cell proliferation.High levels of gal-3 are linked to cancer growth and cancer progressionto a metastatic stage in a stunning variety of cancers. A number ofcancers have been specifically linked to or associated with elevatedgal-3 levels, including liver cancer, kidney cancer, breast cancer,prostate cancer, colon cancer, thyroid cancer, cancer of thegallbladder, nasopharyngeal cancer, lymphocytic leukemia, lung cancer,melanoma, multiple myeloma, glioblastoma multiforme, uterine cancer,ovarian cancer, cervical cancer, brain cancer and others. Elevated gal-3levels have also been shown to interfere with or suppress conventionalantineoplastic regimens, such as chemotherapeutic treatments likecis-platinum, doxorubicin and related chemotherapeutics.

Inflammation is a commonly encountered body condition—a natural responseof the body to a variety of diseases and trauma. As with the otherconditions noted above, gal-3 levels above normal levels are implicatedin a wide variety of situations where harmful inflammation isencountered. Again, the list of conditions and disease states is tooextensive to exhaust every possibility, but inflammatory conditionsassociated with elevated gal-3 levels include aggravated inflammationassociated with non-degradable pathogens, autoimmune reactions,allergies, ionizing radiation exposure, diabetes, heart disease anddysfunction, atherosclerosis, bronchial inflammation, intestinal ulcers,intestinal inflammation of the bowels, cirrhosis-associated hepaticinflammation, parasitic infection associated inflammation, inflammationassociated with viral infection, inflammation associated with fungalinfection, inflammation associated with arthritis, with multiplesclerosis, psoriasis, Alzheimer's disease, Parkinson's disease, andamyotrophic lateral sclerosis (ALS). Again, while inflammation is apathway frequently employed by the body in responding to any number ofchallenges, elevated levels of gal-3 have been found to aggravate theinflammation, causing damage and injury leading to morbidity ormortality in a wide variety of situations that are otherwise manageable,including inflammation due to heavy metal poisoning and similar toxins,stroke and related ischemic injuries, liver inflammation due toacetaminophen, a number of T-cell mediated responses generally involvedin autoimmune diseases and the like. Gal-3 is also involved with kidneyinjury and kidney disease, hepatitis, pulmonary hypertension andfibrosis, diabetes, and gastrointestinal inflammatory conditions such asulcerative colitis, Crohn's disease, celiac disease, and others.

As noted, elevated levels of circulating, active gal-3 are associatedwith, and apparently aggravate, a number of inflammatory conditions,including those contributing to heart, kidney, lung, brain, and liverdisease. Gal-3 is also associated with a fibrotic formation,particularly in response to organ damage. Higher levels of circulatinggal-3 are found to induce pathogenic fibroses in cardiovascular disease,gastroenterological disease, cardiovascular trauma, renal tissue trauma,brain trauma, lung trauma, hepatic tissue trauma, tissue damage due toradiation therapy and diseases and conditions of connective tissue andskin such as systemic sclerosis.

Accordingly, the art is replete with observations that elevated levelsof gal-3, as well as gal-1 and gal-9, can complicate or exacerbate awide variety of disease and injury conditions. It would be of value tofind a way to control inflammation and formation of fibroses, where theinflammation and fibroses are injurious, particularly in theenvironments described above, and notably in cardiac care and otherorgan tissue disease and trauma. By the same token, it would be of valueto control the cellular responses mediated by gal-3 that accelerate cellproliferation and transformation, including the formation and growth oftumors, the transformation of cancer cells and metastatic spread ofcancer. Another goal in the art is to avoid the problem posed by theinterference in the treatment of cancer by conventional agents, likebleomycin, Adriamycin, doxorubicin, cyclophosphamide and cyclosporine.Some of the side effects caused by these agents are gal-3 mediated, andcan be addressed and ameliorated by the invention. Elevated gal-3 levelsalso appear to interfere with pharmaceuticals used in otherapplications, such as the antiarrhythmic drug amiodarone, and statindrugs.

Plasmapheresis is a blood separation technology, where blood is divertedfrom the body through a needle or catheter to a separator which removesblood cells and returns them to the body, leaving plasma. This type oftechnique has been used historically in the treatment of autoimmunediseases, where the antibodies at issue are removed by contacting theplasma with the ligands to which they bind. The plasma is then augmentedas required, with anticoagulants, therapeutics and associated elements,and returned to the body. In prior art methods employing plasma exchangeor replacement therapies generally, as illustrated in U.S. patentpublication US 2006/0129082, the technology was used to target andremove “toxic serum components” such as ammonia, uric acid, and cellgrowth inhibitors. The same reference, at [0009]-[0010] warns againstthe use of plasma exchange in general. Similar warnings are sounded inKyles et al, Am. J. Crit. Care, 14, 109-112 (2005) reviewing the use ofplasmapheresis for support of immunoglobulin sepsis treatment, notingthat traditionally, plasmapheresis has been used in treatments to removepathogenic autoantibodies and endotoxins in autoimmune disorders and toremove harmful substances produced by the infecting organisms causingsepsis. As far as Applicant is aware, this invention reflects the firstuse of plasmapheresis to lower and control the level of active gal-3, anessential and relatively low molecular weight molecule, to supportpatients in specific need of that relief.

An early form of apparatus for plasmapheresis is set forth in U.S. Pat.No. 3,625,212, which describes measures to ensure return of treatedplasma, as well as the separated blood cells, to the proper donor. U.S.Pat. No. 4,531,932 addresses plasmapheresis by centrifugation, themethod used to separate out the red blood cells, on a rapid andnear-continuous basis. U.S. Pat. Nos. 6,245,038 and 6,627,151 eachdescribe a variety of methods of separating out plasma contents andreturning the treated plasma to the patient after first removing redblood cells, in general, to reduce blood viscosity by removal of highmolecular weight protein. While the invention that is the subject ofthis application focuses on the reduction in galectins circulatinglevels, such as gal-3 levels, and not high molecular weight proteins ordirectly addressing viscosity, the disclosure of these four (4) patentsis incorporated herein-by-reference for their disclosure of availableplasmapheresis techniques and apparatus which may generally be employedin this invention. Advances in apheresis generally, includingplasmapheresis, have demonstrated the effectiveness of the use ofhemodialysis equipment using a highly permeable membrane like thePlasmaflo AP-05H from Asahi Medical and a standard dialysis machine inultrafiltration mode. This is similar to hemoperfusion in application.The use of hemodialysis and single needle plasmapheresis are welltolerated, and favored for use in this invention.

Prior to the development of this invention, those of skill in the arthad experimented with the reduction of gal-3 levels in various respects.Thus, the activity of gal-3 in aggravating or promoting cancer, as wellas the ability of a cancer to metastasize, is widely commented on in theliterature following 2006. These literature findings stress repeatedlythe importance of binding or reducing the circulating concentration ortiter of gal-3, and/or inactivating gal-3 through gal-3 binders such asMCP. See, for example, Wang et al, Cell Death and Disease, 1-10 (2010)(gal-3 inhibition promotes treatment) and Yu et al, J. Biol. Chemistry,Vol. 282, 1, pp. 773-781 (2007) establishing that gal-3 interactions mayenhance formation of cancer or transformation of metastatic cancer.

As disclosed and claimed in U.S. Pat. No. 6,274,566, Gal-3 binders suchas MCP and other compounds can bind to circulating tumor cells (CTC's)and prevent them from creating new metastasis. These CTC's are oftenimplicated in mutations and a more aggressive disease. Cancer stem cellsthat may also be circulating and get stimulated under conditions ofstress and inflammation, provide gal-3 another mechanism for aggravatingcancer. The method of these prior cases may be used in conjunction withthe invention of this application. In particular, when there are a highnumber of gal-3 molecules circulating in the blood stream it makes itmore difficult for the gal-3 binders to target these CTCs. In thisrespect, gal-3 molecules serve as decoy molecules. The decoy prevents,in this particular application of the invention, binding of the cancercells in the circulatory or lymph system, as opposed to tissue levelgal-3.

As a consequence, reports link acceleration of cancer formation andtransformation to circulating gal-3 concentrations, and suggest thatreducing gal-3 circulating concentrations, reducing its free expressionor otherwise reducing available gal-3 or gal-3 interactions improvescancer prognosis. Zhao et al, Cancer Res, 69, 6799-6806 (2009), Zhao etal, Molecular Cancer 9, 154, 1-12 (2010) and Wang et al, Am. J. ofPathology, 174, 4, 1515-1523 (2009) wherein siRNA-induced reduction ofgal-3 is shown to slow the course of prostate cancer. Similarly,high-risk bladder cancer recurrence and prognosis is related indirectlyto gal-3 levels. Rodriguez et al, J. Curr. Opin. Urol. 22 (5):415-20(2012) and Raspollini et al, Appl. Immunohistochem. Mol. Morphol. (July2012). Clearly, there is substantial literature that supports theconclusion that reducing circulating gal-3, either by blocking itsexpression, or by binding it, is important in controlling cancer, bothin tissue and in circulation.

Circulating gal-3 is empirically implicated in a wide variety ofbiological conditions, however. Cardiac fibrosis is gaining significantattention as a complicating risk factor in cardiac disease, and inparticular, chronic heart failure (CHF). Lok et al, Clin. Res. Cardiol,99, 323-328 (2010). DeFillipi et al, U.S. Cardiology, 7,1, 3-6 (2010)clearly indicate that circulating gal-3 is an important factor infibrosis of many organs and organ systems, and that reducing circulatinggal-3 may have an important role in remediating cardiac injury andprogression to heart failure (HF). Similarly, Psarras et al, Eur. HeartJ., Apr. 26, 2011 demonstrate that reduction in gal-3 levels in themyocardium may reduce fibrosis in the heart and improve outlook. De Boeret al, Ann. Med., 43,1, 60-68 (2011) identify gal-3 as a key indicatorin cardiac health. Shash et al, Eur J. Heart Fail., 12, 8, 826-32 (2011)identify gal-3 levels as a key agent in heart failure through fibrosis.De Boer et al., Eur. J. Heart Fail., 11, 9, 811-817 (2009) link anincrease in gal-3 expression and presence to heightened fibrosis, andheart failure. The same article links gal-3 to inflammation.Inflammation is the hallmark of arteriosclerosis and therefore gal-3levels also contribute to coronary artery disease, peripheral arterydisease, strokes, and vascular dementia.

Fibrosis and inflammation, both mediated to some degree by gal-3(cellular or circulating) are implicated in a variety of conditions ofthe mammalian body, not just cardiac injury and heart failure. Thebinding of gal-3 achieved by administration of low molecular weightpectins (at least, as reflected in U.S. patent application Ser. No.11/485,955, 10,000-20,000 Daltons molecular weight such as MCP) iseffective in reducing trauma due to kidney injury. Kolatsi-Jannou et al,PlusOne, 6, 4, e18683 (2011). Reducing circulating gal-3 levels may beeffective in reducing fibrosis in the lungs and associated asthma.Cederfur et al, Biochim. Biophys. Acta. 1820 (9):1429-36 (2012). Thereduction in circulating gal-3 levels is also indicated to reduceinflammation associated with type 2 diabetics, and similar metabolicdiseases, as well as obesity. Weigert et al, J. Endocrinol. Metab. 95,3,1404-1411 (2010). Thus, high levels of gal-3 have been linked tothyroid cancer, Sethi et al, J. Exp. Ther. Oncol., 8, 4,341-52 (2010)and reduction of gal-3 expression and circulation may delay or reducetumor cell transformation. Chiu et al, Am J. Pathol. 176, 5, 2067-81(2010).

As noted, gal-3 is implicated in a wide variety of biologicalconditions, and a reduction in gal-3 activity, such as that which can beachieved by gal-3 binding with MCP and similar low molecular weightpectins may be of value in treating gastric ulcerative conditions.Srikanta, Biochimie, 92, 2, 194-203 (2010). Kim et al, Gastroenterology,138, 1035-45 (2010) indicate that reducing gal-3 levels may be oftherapeutic value in reducing gastric cancer progression. By the samemethodology, reducing gal-3 levels sensitizes gastric cancer cells toconventional chemotherapeutic agents. Cheong et al, Cancer Sci., 101, 1,94-102 (2010). Gal-3 is implicated in a wide variety of gastrointestinalconditions. Reducing gal-3, by binding for example, may reduceinflammation in the gut mucosa, making MCP an important agent fortreatment of ulcerative colitis, non-specific colitis and ileitis,Crohn's disease, Celiac disease, and gluten sensitivity. Fowler et al,Cell Microbiol., 81,1,44-54 (2006).

Biliary artesia, a liver disease, is associated with extensive fibrosisof the liver linked with elevated gal-3 levels. Honsawek et al, Eur. J.Pediatr. Surg., April, 2011. Reduction of gal-3 levels resulted in ageneral improvement in hepatic health, including reducing inflammation,hepatocyte injury and fibrosis. Federici et al, J. Heptal., 54, 5,975-83 (2011). See also, Liu et al, World J. Gastroenterol. 14,48,7386-91 (2008) which reported, following Applicant's teaching in 2005and 2006 to administer low molecular weight MCP, that MCP inhibitedliver metastases of colon cancer and reduced gal-3 concentrations. MCP,or other gal-3 binders, may be used for prevention of liverinflammation, liver fibrosis and liver cirrhosis as well as post-diseaseliver damage, including the various viral hepatitis diseases (A, B, C,and others) and may be used as well in the treatment of parasitic andchemical hepatitis, chemical liver damage, and others. Gal-3 levels areimplicated in a wide variety of liver associated ailments. Thus, gal-3may be important in the control of Niemann-Pick disease type C, which isa lysosomal disorder characterized by liver disease and progressiveneurodegeneration. Cluzeau et al, Hum. Mol. Geent. 14; 21 (16) 3632-46(2012). There is increasing evidence that elevated gal-3 levels are tiedto acetaminophen-induced hepatotoxicity and inflammation. Radosavljeciet al, Toxicol. Sci., 127:609-19 (2012). Reduction in gal-3 levels mayimprove treatments. Dragomir et al, Toxicol. Sci. 127 (2):609-19 (2012).

While administration of MCP, or a similar binding agent, continues to bea promising therapy of inhibition of damage, and repair of damage,induced by gal-3, the inventor has continued to work to find othermethods of providing faster or more profound relief It has now beenfound that by selective use of certain gal-3 binding molecules, gal-3and specifically biologically active gal-3 can be specifically removedfrom the plasma in significant amounts. Return of the plasma with areduced titer of active gal-3 offers immediate opportunities for therapyand intervention that may be different from, or more profound than, thereduction achieved by administration of binding molecules to a mammal inneed of same. By removing the circulating gal-3 molecules the inventionremoves these protective but potentially harmful molecules from thecirculation. In addition, it allows targeted gal-3 blockers such as MCP,and possibly other oligo-saccharides and various pharmaceutical agentsto be developed to better attach to the gal-3 on the cell surface and onthe tissue level. As the expression of gal-3 is increased in the injuredand inflamed tissue, such as remodeled cardiac muscle or cancer tissue,by removing the circulating gal-3, the gal-3 binding agent can moreeffectively bind to the gal-3 in the target tissue.

SUMMARY OF THE INVENTION

The invention resides in the removal of biologically active gal-3, aswell as biologically active problematic galectins, such as gal-1 andgal-9, from a mammal's circulation by apheresis of one type or another.The invention is generally applicable to removal of any galectin whichmediates biological phenomena such as autoimmunity and cellproliferation (gal-9 and -1, respectively), such that their removal cansupport or improve therapies already in existence .The mammal may be ahuman, a primate, a model such as a rat or mouse, a commercial animalsuch as a cow or pig or goat, or a companion animal such as a dog orcat. Non-human mammalian animals for treatment include primates, both asmodels and as test beds for treatments and intervention that may benefitfrom removal of gal-3 from circulation. Removal is achieved byplasmapheresis, a process traditionally developed and used to removeantibodies from the circulation of those suffering from autoimmunedisorders and the like.

Apheresis is defined as a procedure in which one of the components ofblood is removed. Plasmapheresis of course addresses removal of plasma.Therapeutic apheresis is a process in which whole blood is removed froma patient and separated into components, thus allowing a single elementto be removed or modified while the remaining components are returned tothe patient. The aim of one form of plasmapheresis, also known astherapeutic plasma exchange (TPE), is to remove a large fraction of thepatient's plasma from the body, and to exchange this with replacementsolutions using automatic devices. This may be the patient's plasmafollowing treatment of some type, such as removal of gal-3 from thatplasma, or a plasma substitute that is galectin free.

TPE differs slightly from donor apheresis, which is used to collectplatelets, granulocytes, or peripheral blood stem cells from normalindividual donors. The volume of blood to be exchanged is based on akinetic model of an isolated one-compartment intravascular space, whichassumes that the component is neither synthesized nor degraded duringthe procedure and that it remains within the intravascular compartment.The time interval between plasma exchanges is generally chosen based onthe need to allow the component of interest to re-equilibrate into theintravascular space and the need to minimize the risk of bleeding as aresult of dilutional coagulopathy. In certain instances such as when thepatient has extremely elevated serum gal-3 levels, in individuals wherethe gal-3 levels rise quickly between TPE intervals, or individuals witha preexisting condition such as viral infections, autoimmune disease,hematologic disorders [hyperviscosity syndrome, cryoglobulinemia,porphyria, sickle-cell anaemia, immune complex disease, cold agglutinindisease, hemolytic uremic syndrome, autoimmunehemolytic anemia,autoimmune thrombocytopenia, autoimmune neutropenia, Clq deficiency, andsecondary immunodeficiency, graft versus host disease (GVHD), etc.], anddrug toxicities as explained, infra, it may be preferred to perform aplasma exchange introducing amounts of exogenous plasma, or exogenousisotonic solutions such as normal saline, with or without albumin andother components, to increase benefits of lowering gal-3 in anexpeditious manner and to reduce complications. Since removing gal-3more rapidly than it can be renewed in the body is of importance, theneed to decrease time between intervals of gal-3 lowering plasmaphersismay also lend itself to more efficacious therapy if the replacementtherapy includes an amount of replacement plasma in the process tomaintain low levels of gal-3 in the serum. With the inclusion of plasmaexchange into the procedure, any circulating pathogens and toxins canalso be reduced in individuals with these concerns.

Plasma exchange allows for removal of other components that havedetrimental effects, and can therefor contribute to the efficacy of thetherapy. For example, immune inhibitory components that circulate in theplasma. Another example are TNF alpha receptors that can be present atelevated amounts in the blood stream of cancer patients.

In TPE, the patient's plasma is removed from the blood by centrifugationor a cell separator/filter and is replaced with saline, albumin, and/orfresh frozen plasma (FFP), thus maintaining volume and oncoticequilibrium. (If it's replaced with FFP it is necessary to pre-screen ortreat the FFP so as to maintain and ensure reduced gal-3 level). Ananti-coagulant is added to the blood to help avoid clotting. Theprocedure must be controlled to ensure that the patient is kept in fluidbalance, maintaining a stable, normal plasma volume. Before each TPE forreducing gal-3 serum levels, results of baseline levels of gal-3, basalcomplete blood count, serum protein electrophoresis, coagulation testsand serum electrolytes must be known. Additional laboratory testing suchas fibrinogen, ESR, PT/PTT (INR) and comprehensive metabolic panel mayalso be used to assess the patient. The efficacy of the therapy andtreatment interval regimen is assessed by these values when compared topost therapy levels. Depending on the separation chamber configurationof automated TPE and whether the blood is batched and processed, orwhether separation, removal, and return are ongoing simultaneously,these systems can be classified as intermittent (also calleddiscontinuous) or continuous flow. As a general rule, high-flowautomated devices (1) are primed to allow for removal of all air fromthe circuit and maintenance of the patient's volume, (2) require venousaccess for inflow to the machine and blood return to the patient, (3)have a significant extracorporeal volume, which approaches 500 mL foradult configurations, and (4) are programmable with patient sex, height,weight, and hematocrit and permit blood to be monitored by a marker(e.g. gal-3).

In plasmapheresis, blood is removed from the patient, and blood cellsare separated out from the plasma. The blood cells are returned to thebody's circulation, diluted with fresh plasma or a substitute.Conventional plasmapheresis methods include medications that can includeblood thinners as necessary. While in typical plasmapheresis, the plasmais run over proteins to which the target antibodies bind, in thisparticular case, the plasma is returned to the blood with theantibodies, cytokines, lymphocytes and other blood components, afterhaving had gal-3 selectively removed or inactivated by contact withgal-3 binding molecules. In the case of autoimmune diseases, the removalof gal-3 from the plasma can be an adjuvant therapy added to thetraditional plasmapheresis performed for such patients. Other cytokinesand plasma components may be removed in the process, based on thespecific condition and the specific individual.

This treatment may be used for all the conditions where galectin,including gal-3, levels are elevated in the blood or serum or whereexpression of gal-3 in the tissues is too high. Tissues will shed excessgal-3 into the blood stream where it can be removed through thisinvention. Treatment can be varied depending on the patient, theseverity of the condition and the rate of the mammalian patient'sexpression of gal-3. Ordinarily, treatment every two to four weeks iscontemplated until the condition is resolved, but treatment may be dailywhere required, or at any frequency there between. Daily treatmentincludes one or more plasma exchange sessions in a given day, orcontinuous plasmapheresis over a multiple hour period in acuteconditions. This may be particularly the case where there has been abuild-up of gal-3 levels in the tissues of the patient being treated.Initial removal of gal-3 from the plasma may cause gal-3 from tissue toshift to vascular compartments rapidly, necessitating repeat treatmentsuntil tissue levels are depleted sufficiently to reach equilibrium.Treatment can be administered on an acute or a chronic basis.Advantageously, this treatment is combined with the administration ofgal-3 blockers and inhibitors, such as disclosed in U.S. patentapplication Ser. No. 13/153,618. Although MCP is a target inhibitor,other gal-3 inhibitors, such as other modified carbohydrates, includinglactulosyl-1-leucine, Zou et al, Carcinogen. (2005), as well asantibodies specific for gal-3, and other antagonists like very lowmolecular weight pectin weighing as low as 1 KD, GCS-100, Demotte et al,Can. Res., 70 (19):476-88 (2010), Streetly et al, Blood, 115(19):3939-48 (published Feb. 26, 2010 as an abstract), may be used. GCSis a polysaccharide derived from MCP, as opposed to reduced MCP. A largevariety of gal-3 binding antibodies are commercially available, fromsuppliers including abcam (ab2473), Novus Biologics (NB 100-91778) andAbgent (AJ13129). Other galectins-3 specific antibodies may be used. Byremoving large levels of plasma active gal-3 from the blood, the diseaseand injury due to inflammation or fibroses may be reduced, and theprogression of cancer may be impeded. Similarly, conventionaltherapeutic treatments may be rendered more effective.

In a preferred embodiment, at the same time active gal-3 is removed,soluble TNF receptors, both R-1 and/or R-2 at different ratios based onthe condition, are removed, through the same process, by running theplasma fluid over a bed of binding agents of TNF receptors. TNF can thendirectly target cancer cells or other targets as an effective treatment.The reduction of active gal-3 in both the circulation and the tissuelevel will allow TNF to exert its beneficial effects with a reducedamount of inflammation and fibrosis which limits its use. Wu et al,Arch. Dermatol. 20:1-7 (2012). The effective removal of serum gal-3 alsoenhances chemotherapy, particularly, but not exclusively, when combinedwith TNF receptor removal. Chemotherapy enhancement will take place byeffective removal of serum gal-3, reducing drug resistance, even if noTNF receptors were removed from the circulation. Yamamato-Sugitani etal, PNAS, 18:108 (42), 178468-73 (2012). Gal-3 interferes withplatinum-based chemotherapy and other anti-cancer agents, and increasescell adhesion, and angiogenesis. Wu et al, Cell Oncol. 35 (3):175-80(2012). In addition, removal of gal-3, by plasmapheresis alone, ortogether with administration of circulating gal-3 binders like lowmolecular weight MCP, may effectively treat the diseases and conditionsaddressed above. In addition, this can be further enhanced by combiningit with other therapies, one example being chemotherapy in cancer.

Typical circulating gal-3 level averages for a Caucasian adult rangefrom 7 on up to about 20 ng/ml, with a value of 12-15 nanograms of gal-3per milliliter of serum being a representative and reported value.Patients at risk, including those with advanced illnesses, exhibitlevels, without treatment, that can be much higher than that patient'saverage or normal level. In accordance with the invention, individualsfacing serious illness or continued disability due to gal-3 mediatedfibrosis, gal-3 mediated inflammation, and cancer growth, transformationand metastases associated with elevated gal-3 levels are treated byplasmapheresis to achieve a significant reduction in circulating gal-3titer.

By significant reduction in circulating gal-3 levels, inflammationand/or fibrosis due to trauma or disease condition can be controlled.Similar reductions in gal-3 levels can aid in the control of the growth,spread and the transformation of various kinds of cancer. In general, areduction of circulating gal-3 of at least ten percent (10%) isnecessary to achieve significant progress in gal-3 mediated fibroses,and even more may be required in acute conditions involvinginflammation, fibroses due to trauma or aggressive cancer. In functionalterms, the reduction of gal-3 should be sufficient to reduce or inhibitthe impact of gal-3 levels on inflammation and fibroses, or cancergrowth and transformation, in said patient. Reduction in circulatinggal-3 of at least twenty percent (20%), and in some cases at least fortypercent (40%) or even fifty percent (50%), may be required on asustained basis. Severe situations may require reduction in circulatinggal-3 levels in a mammalian patient of greater than fifty percent (50%)of that patient's circulating gal-3 titer, on up to seventy-five percent(75%) or even more. While some level of gal-3 in circulation is requiredfor homeostasis, in acute situations, reductions at least by eightypercent (80%) of circulating gal-3, on up to near total removal of gal-3from serum, may be called for, as that level is quickly replenished bythe body. Acute situations can be found in all sorts of individuals, buta representative example is hepatic inflammation or transformation in anaggressive cancer like pancreatic cancer or small cell lung carcinoma.

The gal-3 levels in races other than Caucasians and subjects may vary,but the target is to reduce gal-3 levels below the appropriate normalvalue. Target levels can vary based on the condition, age, gender, andother therapies involved. As a general matter, treatment of the patientaccording to this invention may begin with plasmapheresis in conjunctionwith the absorptive column designed to reduce the patient's gal-3 to apreselected value consistent with good health and homeostasis in thatindividual. In some cases, it may be necessary to repeat or extend thattreatment to achieve even greater reductions.

This invention is straightforward in its application. It is recognizinghow many different indications are served by this technology that iscomplex and startling. In the current invention, blood is removed fromthe patient according to well established protocols generally used forplasmapheresis. See, generally, Samuels et al, editors, Office Practiceof Neurology, 1996. The removed blood is treated to remove blood cellsfrom the plasma. These blood cells, together with an additional volumeof plasma or plasma substitute, are returned directly to the patient. Ina single session, two to four liters of plasma may be removed, filtered,and replaced. The blood can also be recycled and recirculated extracorporally, and filtered as needed, for a number of times (continuously)until the desired reduction in serum levels of galecitn-3 is achieved.Different serum levels can be targeted for different conditions. Theblood cell-depleted plasma is then introduced to a chamber where gal-3is removed or inactivated by binding antagonist, possibly creating apermanent bond that inactivates the gal-3. One of two alternativemeasures may be used to remove gal-3, although they may be combined. Ina first alternative, the plasma is admixed with a particle which bindsgal-3. Preferably, this is an antibody or similar ligand, or apolysaccharide derivative that is most preferably MCP, but any agentthat can bind gal-3 can be used. Methods of preparing low molecularweight pectins are known in the art, and set forth in U.S. patentapplication Ser. No. 13/153,648. Alternate adsorbent galectin affinitycolumns can be prepared with matrix linked compounds having multivalentpresentation of galactose, lactose, poly-N-acetyllactosamine, Nacetyl-D-lactosamine, lacto-N-tetraose, lacto-N-hexosespecific peptides,aptamers (oligonucleic acid or peptide molecules), oligo-saccharides,glycoproteins (such as alpha-2 macroglobulin and haptoglobulin),antibodies, engineered Fc (fragment, crystallizable) and Fab (fragmentantigen binding) antibody fragments, Thomsen-Friedenreich glycoantigen(TFAg) or with small carbohydrate (galactose) derivatives.

The binding agent is modified to be complexed with an agent that iseasily removed. In one embodiment, this is a magnetic particle. Afterproviding for adequate circulation time, a magnetic field is applied tothe fluid comprising the plasma and the MCP complex, and the bound gal-3can be drawn off. Different filters that incorporate gal-3 binders canbe used in the plasmapheresis process.

In certain conditions such as cancer, the circulating gal-3 can beviewed as a sort of decoy released by the cancer cells. It has aprotective quality as it doesn't allow the host, and doesn't allow gal-3binders such as MCP to reach the target tissue where galecin-3 is overexpressed. It also induces inflammation and fibrosis and makes it moredifficult for the host to bind to the gal-3 in the tissue and cellsurface level. Removing the circulating gal-3 provides both atherapeutic treatment on its own and allows other agents to bind andinactivate the gal-3 in the target tissue level. This is similar to TNFAlpha and circulating TNF alpha receptors. Such plasmapheresis can becombined with plasmapheresis of other compounds, and can enhance animmune response and an anti-inflammatory response. The reduction ofcirculating gal-3 will allow one of skill in the art, typically amedical practitioner with at least five (5) years of experience in thefield in addition to appropriate educational experience, to more easilyneutralize and inactivate the tissue expressed gal-3, thus allowing fora local immune response with less inflammation and fibrosis. As such, itcan be combined with removal of TNF Alpha receptors, both R-1 and R-2.It can also be combined with administration of TNF alpha or agents thatenhance TNF alpha activity.

Removing or reducing the level of circulating gal-3 can reduce thesystemic and unwanted inflammatory process, resulting, as demonstratedin the kidney MCP study, with reduction in levels of IL-6, andconsequently TNF alpha and TNF kappa beta.

Accordingly, the invention disclosed herein operates on two (2) levels:

-   1. Direct reduction of circulating gal-3; and-   2. Ability to better target the gal-3 in the tissue level.

This has several consequences in terms of treatment effectiveness:

-   A. By reducing the circulating gal-3, there can be greater shedding    of the tissue gal-3 through greater gradient difference, resulting    in reduced inflammation, fibrosis and remodeling in the tissue    level.-   B. Reduction of secondary pro-inflammatory cytokines such as IL-6,    TNF alpha, TNF kappa beta, and others.-   C. It can allow a greater efficacy of circulating various gal-3    blockers in general, and specifically modified citrus pectin (MCP)    and polyuronides under 40K Dalton.-   D. It can increase the efficacy of other therapies that are    inhibited by excessive circulating gal-3.

In an alternative embodiment, the gal-3 comprising plasma may be runpast a solid phase of immobilized gal-3 binding agents. MCP is oneexample and gal-3 specific antibodies, bound to a column or tube, areanother. In the preferred embodiments, these two approaches to removalof gal-3 from circulation are combined. They can be combined in eitherorder, but running the plasma past an immobile phase, followed bycombining the plasma with an easily removable binding agent ispreferred. Alternately the binding of an antagonist to gal-3 may beadequate to inactivate the molecule, and thus can be returned to thebody without the step to remove it from the plasma.

The binding of gal-3 by a plasmapheresis element that will remove itfrom circulation is an event that will aid medical conditions over awide variety of indications. On a broad scale, the indications areprincipally associated with inhibiting tumor growth and transformation(cancer), inflammation and fibrosis and enhancing innate immunecapacity. These are implicated in specific indications such as, heartdisease, kidney damage, liver damage, hepatic and renal disease, bladderdisease, thyroid disease, pulmonary disease, gastrointestinal disease,immune response, stroke, persistent acute inflammation due tonon-degradable pathogens, persistent foreign bodies, autoimmunereactions, hypersensitivities and allergies, pesticides, environmentaltoxins, and heavy metals, as well as heterogenic conditions such asradiation (examples being medical procedures such as various radiationtherapies, exposure to ionizing radiation, nuclear radiation, cosmicradiation, electromagnetic radiation), chemotherapy damage, and postradiation and chemotherapy induced inflammation and fibrosis,post-surgery rise in inflammation, acute traumas such as accidents,GVHD, and others.

Elevated circulating gal-3 can change a localized situation, such aslocalized inflammation or fibrosis, and convert it into a larger,systemic problem. Thus, when gal-3 binds to components in the blood,which also bind toxic agents and the like, or similarly, when localizedtoxins are bound by gal-3, the damage potentially caused by these agentsproximate to a localized injury or diseased tissue can become systemic.The same phenomenon is observed in connection with potentiallymetastatic cancer. Gal-3 is a generally adhesive molecule. Elevatedgal-3 levels will accelerate the spread of cancer from a localized tumorto a system wide, multi-organ problem. Reducing elevated gal-3 levelsbelow 15 or 12 ng/ml, by ten percent (10%) or more, will help tolocalize injury and damage, and maximize the benefit of unrelatedtherapeutic agents at the local injury or disease site.

As noted above, elevated gal-3 levels are associated with growth,transformation, angiogenesis and metastatic migration of cancer cellsacross a wide variety of cancers, including liver cancer, kidney cancer,breast cancer, prostate cancer, colon cancer, thyroid cancer, cancer ofthe gallbladder, nasopharyngeal cancer, lymphocytic leukemia, lungcancer, melanoma, multiple myeloma, glioblastoma multiforme, uterinecancer, ovarian cancer, cervical cancer, and brain cancer among others,as well as reducing sensitivity in these cancers to conventionalantineoplastic agents.

Elevated gal-3 levels are also associated with the development andextension of fibroses beyond normal and healthy levels, in situationsassociated with cardiovascular disease and heart failure, in tissueinjury including brain, lungs, renal, hepatic, heart andgastroenterological situations as well as tissue damage due to radiationand chemotherapy exposure.

Above-normal gal-3 levels are also encountered in connection withinflammation. This can be disease or trauma associated inflammation, aswell as persistent acute inflammation due to non-degradable pathogens,persistent foreign bodies, or autoimmune reactions, hypersensitivitiesand allergies, ionizing radiation, nuclear radiation and inflammationthat may be associated with disease or organ failure modes, includingdiabetes (I and II), heart disease and dysfunction, atherosclerosis,asthma (bronchial inflammation), gastric and duodenal ulcers, intestinalinflammation in the bowels (inflammatory bowel diseases), hepaticinflammation associated with both alcohol and non-alcohol relatedcirrhosis and inflammation, liver infections such as viral hepatitis,among others. Other indications associated with inflammation andsusceptible to treatment by plasmapheretic treatment to reduce gal-3levels include a variety of parasite-induced conditions, such astrypanosomiasis, cerebral malaria, and inflammation and resistance tovarious infections including Paracoccidiosis brasilensis (fungalinfection), schistosomiasis, granulatomatous bronchopneumonia, Lymedisease, tubercolosis, etc. Reports of elevated gal-3 levels inconnection with infection include Candida albicans, Reales-Calderon etal, J. Proteomics, 3:75 (15) 4734-46 (2012), Schistoma mansoni (aparasitic infection) Brand et al, Histol. Histopathol., 27 (8) 1109-20(2012) and many others, including bacterial infections like Neisseriameningitides, Quattroni et al, Cell Microbiol., (July 2012). Prioninfection, in CNS disease, has also been linked to gal-3 elevatedlevels. Mok et al, Biochem. Bophys. Res. Commun. 3:359:672-8 (2007).Elevated gal-3 levels are an important contributing factor ininflammation associated with arthritis, multiple sclerosis, Parkinson's,other neurological ailments; and other diseases of the skeletomuscularand skin systems, including inflammation and fibrosis related conditionssuch as psoriasis and aging of the skin. See, for instance, Ezzat et al,Int. J. Rheum. Dis., 14 (4):345-52 (2011) (arthritis), Gal et al, Acta.Histochem. Cytochem., 44 (5):191-9 (2011) and Liu et al, Invest.Dermatol., 10.1038 (2012) (wound healing) and Larsen et al, Dermatol.Sci., 64 (2):85-91 (2011) (skin diseases). As noted above, theseconditions may be treated by removal of biologically active, unboundgal-3 from circulation by this invention alone, or by removal fromcirculation combined with administration of gal-3 binding agents such asMCP to further address gal-3 mediated conditions.

Gal-3 has been shown in multiple studies to contribute to the ability oftumors to evade the immune system. This can occur via multiplemechanisms: Tumor cells, secrete gal-3 into the tumor micro environmentwhere the gal-3 via its self-adherent properties has a cloaking effecton cancer cells, effectively preventing immune cells from interactionwith the antigens present on the surface of tumor cells. These immunecells, including CD4 and CD8 tumor infiltrating T lymphocytes, as wellas macrophages, are thus suppressed in their capacity to be activated bytumor cell surface antigens; Immune suppression can occur because gal-3also binds directly to immune cells (lymphocytes and macrophages) ortraps and effectively immobilizes them in a glycoprotein matrix in thetumor microenvironment. These immune cells are found in the tumormicroenvironment in response to antigens that are present on the surfaceof tumor cells. These immune cells, when properly stimulated, secrete anumber of factors, such as cytokines, which are cytotoxic to tumorcells. An elevated gal-3 level in the tumor microenvironment disablestheir ability to activate in response to antigens present on tumor cellsurface. Demotte N. et al, Cancer Res. 70 (19):7476-88 (2010); van derBruggen P., Bull Mem Acad R Med Belg. 164 (5-6):183-91 (2009). Similarprocess allows for different infectious agents to evade the immunesystem, and “hide” from different therapies such as antibiotic therapy.This phenomenon is referred to, in popular terms as Biofilm, and gal-3is an integral part of the biofilm. This may be of specific importancein chronic infections such as Lyme, Bartobela, Babisia, rickettsia, andother co infections, as well as different parasitic and fungalinfections.

Activation of the immune system by reduction of gal-3 is a furtheraspect of the invention. Inhibition of gal-3 has been found to enhancethe proliferation of immune cells in response to antigens presented ontumor cells. Demotte N, et al, Immunity. 28 (3):414-24 (2008). Rapidsystemic removal of gal-3 could be used in conjunction with oral gal-3antagonists to exponentially enhance the effect of freeing the immunesystem to effectively act on tumor cells. This enhanced response viasystemic removal of gal-3 can be used in conjunction with a number ofimmune enhancing therapies currently available or being developed.

Galectins have been documented as main regulators of immune cellhomeostasis and inflammatory processes. Among these, gal-3 with itsanti-apoptotic activity has been reported that increased gal-3expression correlates with defective T-cell apoptosis in patients withsome immune disorders. The level of gal-3 in patients receivingallogeneic hematopoietic stem transplantation (HSCT) in the context ofthe presence of acute graft-versus-host disease (GVHD) has beenevaluated. The findings showed the level of gal-3 was higher in patientswith acute GVHD than those without after HSCT, and also higher aftertransplantation than before or at transplantation day in these patients.These findings suggests that the gal-3 might be one of significantmolecules in pathogenesis of acute GVHD, and the successive evaluationof gal-3 levels might be one of informative tests predicting theoccurrence of acute GVHD. Min Y H et al, Increased level of gal-3 inpatients with acute graft-versus-host disease after allogeneichaematopoietic stem cell transplantation. 31st Annual Meeting of theEuropean Group for Blood and Marrow Transplantation 21st Meeting of theEBMT Nurses Group 4th Meeting of the EBMT Data Management Group, Prague,CZ (2005).

Multiple studies have contributed to the understanding of theimmunosuppressive mechanisms used by mesenchymal stromal cells (MSC).Galectins have recently been discovered as a main regulator of MSCimmunosuppressive function. It has been identified that gal-3 as thefirst human lectin involved in the modulation of the immunosuppressivepotential of mesenchymal stem cells (MSC). The double knockdown ofgalectins-1 and -3 genes have been shown to almost abolish theimmunosuppressive capacity of MSC. The use of a competitive inhibitorfor galectin binding restored alloresponsiveness, implying anextracellular mechanism of action of galectins. The published datademonstrate the involvement of secreted gal-1 and -3 in MSC-mediated Tcell suppression. The immunosuppression by MSC-secreted galectins shouldfacilitate further understanding of the inflammatory reactions such asthose seen in GVHD and autoimmune disorders. Sioud M et al, Int J Oncol.38 (2):385-90. (2011). In particular, allogenic transplants frequentlygive rise to issues involving inflammatory disorders that are mediatedby Gal-3. Gal-1 and gal-3 are constitutively expressed and secreted byhuman bone marrow MSC Inhibition of gal-1 and gal-3 gene expression hascancelled the suppressive effect of MSC on allogeneic T cells. Thisincrease in the understanding of MSC suppressor mechanisms offers aninsight into the use of these cells in human therapy such as thetreatment of GVHD, a severe complication after haematopoietic stem celltransplantation. Sioud M. Scand J Immunol. 73 (2):79-84. (2011). Thus,as in other areas and applications, this invention lends itself toimproving the effectiveness of other therapies by reducing gal-1 andgal-3 mediated reactions that would otherwise interfere with theeffectiveness, in this case, of transplant therapies.

Inflammation mediated at least in part by circulating gal-3 levels alsoplays a role in organic psychiatric and brain disorders. This kind ofinflammation has been associated with a wide variety of conditions, suchas schizophrenia. Muller et al., Adv. Protein Chem Struct. Biol., 88,49-68 (2012) and Palladino et al, J. Neuroinflammation, 22;9, 206(2012). Thus, reducing elevated gal-3 levels may be one method to assistin the control of psychiatric disorders of this type which are difficultto control by therapeutic intervention alone. Similarly, a conditionreceiving increasing attention, attention deficit hyperactivity disorder(ADHD) has been shown to be mediated to some degree by gal-3 expression.Wu et al, Brain Pathol., 20 (6), 1042-54 (2010). Elevated gal-3expression levels, and the inflammation associated therewith, have alsobeen linked to organic tissue damage, as well as psychiatric behavioraldisorders. Thus, Alzheimer's disease and enhanced Aβ amyloid depositshave been shown to be associated with pro-inflammatory conditions, suchas those mediated by elevated gal-3 levels. Reale, et al, Curr.Alzheimer Res. 9 (4), 447-57 (2012). Gal-3 has also been shown to beinvolved in the proper differentiation of oligodendrocytes controllingmyelin sheath conditions, Pasquin et al, Cell Death Differ., 18 (11),1746-56 (2012) and recovery and regrowth following traumatic braininjury. Venkatesan et al, J. Neuroinflammation 27 (7) 32 (2010). Thus,in addition to being of importance in the control of inflammation indisease or injury conditions generally, reduction of circulating gal-3levels through plasmapheresis may be of critical value in controllingfor physical phenomena associated with disorders of the brain andcentral nervous system.

It should be noted that commonly, inflammation and fibrosis can beinduced by deliberate treatment, not just trauma or disease condition.The removal of circulating, unbound gal-3 through this invention can beeffective in reducing or preventing organ damage induced by chemotherapyand other pharmaceuticals. Some examples include bleomycin, whichinduces lung fibroses, and a wide variety of cardiac drugs such asamiodarone. Adriamycin and doxorubicin are widely prescribed and presentcardiac inflammation and fibroses issues. Bacillus Calmette-Guerinwashes to treat bladder cancer induce systemic inflammation andcyclophosphamide also induces bladder damage. Cyclosporine, a widelyused immunosuppressant drug, and the active agent in Restasis™, induceskidney toxicity and inflammation. Studies indicate that the vast arrayof organ damage caused by prescribed pharmaceuticals is mediated, atleast in part, by elevated gal-3 levels, and can be limited if noteliminated by the method of this invention.

Among other specific indications that can be addressed by this inventionare non-alcoholic steatohepatitis (NASH); Sepsis: (See Bibhuti et al,2013); post-trauma nerve regeneration (Narcisco et al, 2009) (Doverhaget al, 2010; delayed xenograft rejection (DXR) Jin, 2006; Chronicallograft injury kidney transplant (Dang et al, 2012);Ischemic-reperfusion injury: (IRI) (Fernandes et al, 2008); Ideopathicpulmonary fibrosis (IPF); GVHD, and related indications.

The invention disclosed herein, the use of at least partialdonor-provided apheresis to reduce active Gal-3 levels in a patient mayfind particular application in the treatment of chronic kidney infectionand end stage renal disease. Currently, there are approximately 26million adults with CKD and 800,000 individuals with Stage 4 CKD and thenumber of these patients is expected to double by 2020. Patients withStage 4 CKD have a ˜10% death rate and the first-year death rate forESRD patients is ˜25%.

In a preferred embodiment, the serum, after having circulating gal-3reduced or removed, as described, is further treated before returning itto the patient's blood stream. Specifically, agents that may be moreeffective in the absence of, or in the presence of reduced levels of,galectin-3 are specifically added. This includes a wide variety ofactive agents, but specifically includes agents such as chemotherapeuticdrugs, immune enhancing therapies, antibiotics and anti microbials ingeneral (anti virals, anti helmetic, anti parasitic) and therapeuticagents for the various conditions. For example, an anti-inflammatorywill work better, cardiac medications, any drugs delivered to address anissue where gal-3 is a contributing factor, or prevents effectivedelivery to the target tissue, will be enhanced by this process. Theseagents will then have the opportunity to work under an environment oflower levels of gal-3. Even if just for a few hours, they can exhibitfull biological activity. Once inflammation, for example, is reduced,naturally less gal-3 is being produced and expressed by the targettissue resulting in lower circulating gal-3 on a long term basis.

Thus, while in one alternative, the invention involves long term orchronic plasmapheresis to maintain reduced gal-3 levels, the inventionalso contemplates intervention on a short term basis, both removingcirculating gal-3 and providing agents otherwise inhibited by gal-3, toswiftly address inflammation in particular. Gal-3 levels can spike as atransient event, in response to trauma for example, having a techniqueto rapidly lower gal-3 levels in the patient, coupled withadministration of active agents that are ordinarily inhibited to somedegree by high levels of gal-3, can offer a lifesaving technique. Inaddition, reducing Gal-3 levels as a means to reduce inflammation canallow other therapeutic agents, an example being an anti microbial inacute infections such as sepsis, or others, to have a better therapeuticresponse, resulting in a life saving outcome.

Although Applicant does not wish to be confined to these few examples, alarge number of conditions have been shown to be mediated by unboundgal-3, such that its removal, by the invention addressed herein, willaid in treatment. It has been demonstrated that reducing free gal-3 inhumans can prevent renal fibrosis and inflammation following kidneyinjury. Both thyroid cancer and lung cancer treatment has beendemonstrated to effectively improve by reducing gal-3 concentrations.Enhanced sensitivity to both radiation and chemotherapeutic interventionmay be achieved by reducing circulating levels of active gal-3 throughthis invention.

Asthma, and related conditions primarily marked by exaggeratedinflammation may be avoided or suppressed by removing circulating gal-3through the process of this invention. These include inflammation of thegastrointestinal tract, and inflammation and the development of fibrosesof the liver, interstitial cystitis, inflammation associated with brainand cognitive function, and others. Inflammation associated withparasite invasion may also be controlled by removal of gal-3, orreducing its circulating level through this invention. Otherinflammation-associated diseases, such as diabetes and arthritis aresimilarly treated. These conditions may ideally be targets of thisinvention as well as administration of circulating gal-3 binding agentslike MCP, and unrelated therapeutic agents.

While the present invention has been disclosed both generically, andwith reference to specific alternatives, those alternatives are notintended to be limiting unless reflected in the claims set forth below.The invention is limited only by the provisions of the claims, and theirequivalents, as would be recognized by one of skill in the art to whichthis application is directed.

What is claimed is:
 1. A method of treating a condition in a mammalmediated at least in part by active gal-3 in the blood, comprising:selecting a mammal in need of reduction of circulating levels of gal-3;and conducting plasmapheresis on the body fluid of said mammal to reducecirculating levels of active gal-3 such that at least ten percent of thecirculating gal-3 in the serum of that individual is removed by saidplasmapheresis and wherein said plasmapheresis is, at least in part,donor apheresis, wherein certain blood components returned to the mammalare obtained from donor mammals.
 2. The method of claim 1, wherein saidmethod further comprises periodically monitoring the level ofcirculating gal-3 in said mammal, and repeating said plasmapheresis onsaid mammal when said level of gal-3 is above a preselected level ofgal-3 for said individual consistent with that individual's gal-3mediated disease state.
 3. The method of claim 1, wherein thecirculating level of galectin-3 in said mammal is reduced by at leasttwenty-five percent (25%).
 4. The method of claim 1, wherein thecirculating level of gal-3 is reduced by said plasmapheresis by aboutfifty percent (50%) of the level of gal-3 prior to said plasmapheresis.5. The method of claim 1, wherein said individual's circulating gal-3level is reduced by said method by at least seventy-five percent (75%).6. The method of claim 1, wherein said step of conducting plasmapheresiscomprises diverting a portion of said mammal's blood from said mammal'sbody to provide a volume of separated blood, removing red blood cellsfrom said separated blood to provide separated plasma and returning saidred blood cells to said mammal's circulation, contacting said separatedplasma with moieties that bind gal-3, and separating out any suchmoieties and gal-3 bound by them from the rest of said separated plasmato provide a plasma with reduced levels of galectins-3, and returningsaid separated plasma with reduced levels of galectins-3 to saidmammal's circulation together with blood components obtained from saiddonor mammals.
 7. The method of claim 6 wherein said moieties compriseantibodies which bind gal-3, carbohydrates which bind gal-3 andcombinations thereof.
 8. The method of claim 6, wherein said moietiesare conjugated to an element that makes separation of said moiety andany gal-3 bound thereby from said separated plasma easier.
 9. The methodof claim 8, wherein said element is a column to which said moieties areaffixed, a scaffold on which an array of said moieties is affixed,magnetically attractable particles, and mixtures thereof.
 10. The methodof claim 3, wherein, upon return of said separated plasma to saidmammal, the level of circulating gal-3 in said mammal is below 15 ng/ml.11. The method of claim 1, wherein said mammal is selected because it isin need of inhibition of the growth or spread of cancer mediated atleast in part by gal-3.
 12. The method of claim 11 wherein said canceris liver cancer, kidney cancer, breast cancer, prostate cancer, coloncancer, thyroid cancer, cancer of the gallbladder, nasopharyngealcancer, lymphocytic leukemia, lung cancer, melanoma, multiple myeloma,glioblastoma multiforme, uterine cancer, ovarian cancer, cervicalcancer, or brain cancer.
 13. The method of claim 11, wherein said mammalis receiving antineoplastic chemotherapy for cancer which therapy isinhibited by gal-3.
 14. The method of claim 1, wherein said mammal isselected because it is in need of inhibition of development or extensionof fibroses mediated by gal-3.
 15. The method of claim 14, wherein saidfibroses are associated with cardiovascular disease, brain disease,gastroenterological disease, cardiovascular trauma, brain trauma, lungtrauma, renal tissue trauma, hepatic tissue trauma, tissue damage due toradiation therapy and tissue damage due to chemotherapy.
 16. The methodof claim 1, wherein said patient is selected because it is in need ofinhibition of inflammation mediated by gal-3.
 17. The method of claim11, wherein said inflammation is associated with non-degradablepathogens, autoimmune reactions, allergies, ionizing radiation, nuclearirradiation, diabetes, heart disease and dysfunction, atherosclerosis,bronchial inflammation, intestinal ulcers, intestinal inflammation ofthe bowels, hepatic inflammation, cirrhosis-associated hepaticinflammation, inflammation associated with parasitic infection,inflammation associated with viral infection, inflammation associatedwith fungal infection, inflammation associated with bacterial infection,arthritis associated inflammation, inflammation associated with organicpsychiatric or brain disorders, multiple sclerosis, and psoriasis. 18.The method of claim 16, wherein said inflammation is due topharmaceutical therapy.
 19. The method of claim 18, wherein saidpharmaceutical comprises bleomycin, amiodarone, Adriamycin, doxorubicin,cyclophosphamide and cyclosporine.
 20. The method of claim 1, whereinsaid mammal is selected as in need of reduction of gal-3 levels so as toimprove the effectiveness of a pharmaceutical to be administered to saidindividual.
 21. The method of claim 20, wherein said pharmaceutical is astatin, an antineoplastic chemical agent, an anti-inflammatory agent, aTNF blocker, or a TNFα activity promoter.
 22. The method of claim 1,wherein before said serum returned to said patient it is admixed with apharmaceutical agent whose activity is improved where galectins-3 levelsare reduced.
 23. The method of claim 22, wherein said pharmaceuticalagent is an antineoplastic agent or an anti-inflammatory agent.
 24. Themethod of claim 1, wherein said mammal is selected as in need ofreduction of Gal-3 levels so as to address graft versus host disease(GVHD) exhibited by said mammal following an allogenic transplant.